论文标题:刺五加水提物抗炎作用及其机制研究
Studies on Anti-inflammatory Effects of Aqueous Extract of Acanthopanax Senticosus and Its Mechanisms of Action
论文作者
论文导师 徐永平,论文学位 博士,论文专业 生物化工
论文单位 大连理工大学,点击次数 614,论文页数 119页File Size8521K
2007-07-01论文网 http://www.lw23.com/lunwen_149772/
aqueous extract of Acanthopanax senticosus;; anti-inflammatory;; macrophage;; inflammatory mediators;; endotoxic shock
刺五加在我国作为一种传统药物应用已有悠久历史。目前，刺五加在国内外主要应用于冠心病、心绞痛、缺血性中风、糖尿病和高血压等疾病的治疗，而在刺五加抗炎作用及机制方面鲜见报道。本论文采用小鼠巨噬细胞炎症模型和小鼠内毒素休克模型，研究了刺五加水提物抗炎作用及其机制。具体实验内容与结果如下： 1．刺五加水提物分别与活化的RAW264.7小鼠巨噬细胞和原代培养的小鼠腹腔巨噬细胞共同培养，采用Griess试剂、NBT还原法、酚红氧化法、ELISA方法和中性红吞噬法检测了巨噬细胞培养上清液中NO、O_2~-、H_2O_2和TNF-α的含量及细胞活力。实验结果显示，刺五加水提物在10～1000μg／mL范围内显著抑制了LPS&IFN-γ诱导产生的NO，ZYM诱导产生的O_2~-和PMA诱导产生的H_2O_2，对LPS&IFN-γ诱导产生的TNF-α无显著影响，细胞活力检测实验表明这些抑制作用不是细胞毒性作用的结果。为了在整体动物水平上证实刺五加水提物对小鼠巨噬细胞产生炎症介质的抑制作用，我们给小鼠腹腔注射刺五加水提物，分离腹腔巨噬细胞，分别检测巨噬细胞培养上清液中NO、O_2~-、H_2O_2及TNF-α的含量。实验结果显示，100和200 mg／kg剂量的刺五加水提物明显抑制了小鼠腹腔巨噬细胞在LPS&IFN-γ诱导刺激下产生的NO，ZYM诱导刺激下产生的O_2~-和PMA诱导刺激下产生的H_2O_2，对LPS&IFN-γ诱导产生的TNF-α无显著影响。上述结果提示刺五加水提物可能通过抑制活化巨噬细胞产生的炎症介质发挥其抗炎功效。 2．NO在炎症发生和发展过程中有着重要的作用，刺五加水提物明显抑制了NO的产生，因此我们进一步深入探讨了刺五加水提物抑制NO产生的作用机制。分别采用Real-Time RT-PCR方法检测iNOS mRNA水平，Western blotting方法检测iNOS、NF-κBp65和NF-κB抑制蛋白I-κBα蛋白的表达，ELISA方法检测NF-κB p65与DNA的结合活性和流式细胞技术检测胞内ROS的含量。实验结果显示，刺五加水提物抑制了RAW264.7小鼠巨噬细胞在LPS&IFN-γ诱导刺激下，iNOS的mRNA和蛋白表达水平，NF-κB p65的核转位、I-κBα的降解，以及NF-κB p65与DNA的结合活性和胞内ROS的产生。表明刺五加水提物通过阻断NF-κB细胞信号通路的活化，抑制iNOS的基因表达，最终抑制了NO的产生。 3．我们建立了LPS／D-GalN诱发的小鼠内毒素休克模型，研究刺五加水提物对小鼠内毒素休克的保护作用及其机制。存活率观察结果显示，100、200和400 mg／kg剂量的刺五加水提物预防组显著提高了小鼠的存活率，而200、400和800 mg／kg剂量的刺五加水提物治疗组小鼠的存活率无明显提高，提示刺五加水提物对内毒素休克有预防作用。我们进一步检测了刺五加预防组小鼠血清和肝脏中相关炎症介质水平，研究刺五加水提物对内毒素休克的预防保护机制。采用Griess试剂检测血清中NO的含量；ELISA方法检测血清和肝脏中TNF-α和IL-10的含量；Western blotting方法检测肝脏中iNOS的表达水平；ELISA方法检测NF-κB p65与DNA的结合活性；HE染色检测主要器官的病理情况。实验结果显示，刺五加水提物显著抑制了血清和肝脏中TNF-α和NO的产生，以及肝脏中iNOS的表达和NF-κB p65与DNA的结合活性，提高了血清和肝脏中IL-10水平，减轻了心脏、肝脏和肺主要器官炎性细胞浸润，病理损伤情况明显改善。表明刺五加水提物通过提高IL-10水平和抑制NF-κB的活性，降低了TNF-α和NO的释放，减轻了重要脏器的炎症损伤，从而发挥其对内毒素休克的保护作用。 综上所述，本研究证实了刺五加水提物对巨噬细胞炎症介质具有抑制作用以及对内毒素休克具有保护作用，并阐述了其抗炎作用机制，为刺五加在炎症疾病的临床治疗及深入研究奠定了基础。
Acanthopanax senticosus (A. senticosus) is known for thousands of years as a medicinal herb and has been clinically used to treat coronary heart disease, angina, ischemic stroke, diabete and hypertension. However, little is known about anti-inflammatory effects and its mechnisms of action. This study aims to investigate the anti-inflammatory effects of aqueous extract of A. senticosus and its mechnisms of action and provide the evidence for the clinical use. In this study, we established the mouse macrophage inflammation model in vitro and mouse endotoxic shock in vivo and investigated the effect and mechanism of aqueous extract of A. senticosus on inflammatory mediators production by mouse macrophages and its protective effect and mechanism against endotoxic shock in mice. 1. We examined the effects of aqueous extract of A. senticosus on NO, O_2~-, H_2O_2 and TNF-αproduction in activated RAW 264.7 macrophages and mouse peritoneal macrophages. The contents of NO, O_2~-、H_2O_2 and TNF-αin the medium supematant, respectively, were measured by using Griess Reagent, NBT reduction, phenol red oxidation and ELISA. In vitro exposure of RAW 264.7 macrophages or mouse peritoneal macrophages to aqueous extract of A. senticosus (10～1000μg/mL) significantly suppressed NO production induced by LPS plus IFN-γ, O_2~- production induced by ZYM and H_2O_2 production by PMA. Then, LPS plus IFN-γ-induced TNF-αproduction wasn"t affected in RAW 264.7 macropahges and mouse peritoneal macrophages with aqueous extract of A. senticosus incubation. Exposure to aqueous extract of A. senticosus had no effect on cell viability. In vivo administration of aqueous extract of A. senticosus (100 or 200 mg/kg, i.p.) to mice significantly inhibited the production of LPS plus IFN-γ-induced NO, ZYM-induced O_2~- and PMA-induced H_2O_2 by isolated mouse peritoneal macrophages ex vivo. Then, TNF-αproduction wasn"t affected in isolated mouse peritoneal macrophages. These results suggest aqueous extract of A. senticosus has an anti-inflammatory effect based on the inhibition of the inflammatory mediators. 2. We examined the effects of aqueous extract of A. senticosus on iNOS gene expression and NF-κB signal pathway by LPS plus IFN-γ-stimulated RAW 264.7 macrophages. Real-Time RT-PCR, Western blotting, ELISA and Flow Cytometry, respectively, were used to measure mRNA level, protein level, NF-κB activation and intracellular ROS content. Aqueous extract of A. senticosus inhibited mRNA and protein level in LPS plus IFN-γ-treated RAW264.7 macrophages. Aqueous extract of A. senticosus inhibited the LPS plus IFN-γ, mediated increase in intracellular ROS production, and this inhibition caused the reduction of the activation of NF-κB. This effect was mediated through the suppression of NF-κB p65 nuclear translocation, DNA binding activity of NF-κB and I-κBαdegradation. These results suggest aqueous extract of A. senticosus suppresses iNOS gene expression and NO production through the inhibition of NF-κB activation. 3. We examined the protective effect and inflammatory mediators regulatory profiles in mice endotoxic shock, which was induced in mice using an intraperitoneal injection LPS and D-GalN simultaneously. NO concentration, the contents of TNF-αand IL-10 and NF-kB activation, iNOS protein level and histoloty of tissues, respectively, were analyzed by using Griess Reagent, ELISA, Western blotting and HE staining. Intraperitoneal injection of aqueous extract of A. senticosus (100, 200 or 400 mg/kg) prior to LPS/D-GalN challenge significantly improved survival rate of mice compared with that of the LPS/D-GalN alone group. In contrast, treatment of aqueous extract of A. senticosus (200, 400, or 800 mg/kg, i.p.) after LPS/D-GalN didn"t reduce the mortality of mice. It is therefore suggested that aqueous extract of A. senticosus exerted prophylactic effect on endotoxic shock in mice. Aqueous extract of A. senticosus preatreament inhibited the production of circulating TNF-αand NO, markedly enhanced that of circulating IL-10. Furthermore, TNF-αand iNOS protein levels in liver were decreased as well as IL-10 level in liver was increased in aqueous extract of A. senticosus preatreated mice compared with the LPS/D-GalN alone group. Aqueous extract of A. senticosus pretreatment inhibited the activation of NF-kB in liver tissue. Inflammatory cells infiltration in heart, liver and lung tissues were alleviated. These results suggest that aqueous extract of A. senticosus protects against LPS/D-GalN induced mice endotoxic shock by a mechanism involving enhancement of IL-10 and inhibition of NF-kB activation, which caused down-regulation of TNF-αand NO, reduction inflammatory cells infiltraion and protection against heart, liver and lung injury. Taken together, our results show the inhibitory effect of aqueous extract of A. senticosus on inflammatory mediators production by mouse macrophages and the prophylactic effect against mice endotoxic shock. Furthermore, its mechanisms of action were elucidated. These data provide the academic evidence for the clinical use and further development of this medicinal herb.
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